Shiga toxin-producing Escherichia coli (STEC) are a group of prevalent foodborne pathogens responsible for outbreaks of human gastrointestinal disease. The morbidity and mortality associated with these outbreaks have highlighted the threat these organisms pose to public health (Karch et al., Int'l J Med Microbiol, (2005) 295:405-18; Gyles, J. Anim Sci, (2007) 85:E45-62; Manning et al., Emerg Infect Dis, (2007) 13:318-21). Most STEC outbreaks have been traced worldwide to the consumption of bacterial-contaminated food. Ruminants are the main reservoir for STEC strains and food contaminated with bovine feces has been linked to severe complications, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (Hussein, J Anim Sci, (2007) 85: E63-72).
STEC possess a number of virulence factors, but Shiga toxins (Stxs) were considered the most critical in disease pathogenesis and are responsible for HC and HUS. Stxs are AB5 holotoxins and are comprised of one A subunit (32 kDa) and five B subunits (7.7 kDa) (Fraser et al., Nat Struct Biol, (1994) 1:59-64; Fraser et al., J Biol Chem (2004) 279:27511-17). The Stx A subunit is an enzymatically active N-glycosidase that inhibits the activity of rRNA by cleavage of an adenine base from the 28S rRNA component of the eukaryotic ribosomal 60S subunit, causing protein synthesis to cease resulting in cell death (Endo and Tsurugi, J Biol Chem, (1988) 263:8735-9). The Stx B subunit is responsible for binding to host cells through interaction with globotriaosylceramide (Gb3) or globotetraosylceramide (Gb4) receptors present on the surfaces of cells (Lingwood, Adv Lipid Res (1993) 25:189-211), leading to subsequent internalization of the toxin. There are two serologically distinct groups of Stxs, Stx1 and Stx2. Recent epidemiological and molecular typing studies suggested that STEC strains expressing Stx2 were more virulent than strains expressing either Stx1 or both Stx1 and Stx2 (Ostroff et al., J Infect Dis, (1989) 160:994-8; Boerlin et al., J Clin Microbiol. (1999) 37:497-503). A mean lethal dose (LD50) for Stx2 of 50 ng/kg in mice was reported by Tesh et al. (Infect Immun, (1993) 61:3392-402) and Lindgren et al. (Infect Immun, (2003) 69:623-31). In contrast to Stx1, many variants of Stx2 have been identified (Weinstein et al., J Bacteriol, (1988) 170:4223-30; Piérard et al., J Clin Microbiol (1998) 36:3317-22; Bertin et al., J Clin Microbiol, (2001) 39:3060-5; Leung et al., Appl Environ Microbiol, (2003) 69:7549-53; Strauch et al., Infect Immun, (1994) 40:338-43). These variants differ from each other in terms of their affinity for host receptors, cytotoxicity, and pathogenicity.
The capacity to control STEC disease in humans and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection. Although the role of Stx2 in these outbreaks has received considerable attention, rapid, sensitive and specific detection methods for this toxin in food are still limited. This is because detection of Stxs in food samples is often difficult due to the combination of low toxin concentration and effect of the complex matrix present in food. Historically, the Vero cell cytotoxicity assay has played an important role in establishing a diagnosis of STEC infection and it still remains the “gold standard” for Stx activity. However, like most activity-based assays, such as the mouse bioassays, radioactivity assays, and cell-free translation assays, the Vero cell assay is time-consuming, requires cell culture facilities, and expensive equipment that is usually not available in many laboratories. Furthermore, a subsequent antibody-based neutralization bioassay is required in order to confirm the presence of the toxin. Other assays, such as receptor-based assays are less time-consuming and enable the discrimination of different toxins, but detailed evaluation and optimization are needed to establish these methods as analytical tools (Uzawa et al., ChemBioChem, (2007) 61:3392-402).
Over the past decades, a number of immunoassays have been developed, the most common ones being the enzyme-linked immunosorbent assays (ELISA). These assays provide multiple benefits. Notably, they are simple, rapid, cost-effective, and all reagents and equipment needed are available in most laboratories. However, the sensitivity and specificity of immunoassays is largely dependent on the quality of the antibodies used. Our recent studies on detecting botulinum neurotoxin type A in milk demonstrated that simple immunoassay formats can be highly sensitive when high-affinity antibodies are incorporated (Stanker et al., J Immunol Methods, (2008) 336:1-8). While antibodies against Stx2 have been described in the scientific literature, few are commercially available. Their expense and lack of sufficient binding affinity to the native toxins make studies focused on constructing a sensitive immunoassay difficult.